Project Summary/Abstract This proposal seeks to develop and validate a novel technology for detecting malignant cells in biospecimen samples of oral and oropharyngeal cancer. This technology will allow screening, diagnosis, and quantification at higher precision and lower cost than what is currently available. In the US, both the incidence and societal impact of oropharyngeal cancer are increasing, despite the marked decrease in use of tobacco products. This is exacerbated by the fact that almost two-thirds of patients have lymph node or distant metastases at the time of diagnosis, dramatically reducing five-year survival rates. In many developing countries, the incidence of oropharyngeal cancer is not just higher but also takes a much more dramatic toll. One key reason for the often late diagnosis of oropharyngeal cancers despite their superficial location is that these tumors typically remain asymptotic, and early lesions are erroneously considered benign. The current standard of care for detecting oral and oropharyngeal cancer remains histopathology, which is labor-intensive and requires highly trained personnel and a sophisticated infrastructure. Thus, a novel tool to quickly, effortlessly, and quantitatively determine the presence of these malignancies is urgently needed. PARPi-FL ? a fluorescent tracer that has been shown to specifically detect PARP1, a highly expressed biomarker for malignant growth ? has been translated to the clinic at Memorial Sloan Kettering Cancer Center (MSK) as an intraoperative imaging agent for oral cancer. However, Summit Biomedical Imaging, LLC (SBI) believes that the non-toxic small molecule could be much more useful as an ex vivo stain for suspicious biopsy and brush cytology samples; thus, MSK is transferring this technology to SBI for further development. The agent is inexpensive and, importantly, stable at room temperature for years and stainings can be completed in less than 10 minutes, which makes it ideal for an off-the-shelf diagnostic test. To generate sufficient evidence for the clinical value of PARPi-FL, both of our Specific Aims will explore a distinct ex vivo application. In Specific Aim 1, SBI will develop a one-step kit, PARP Cytostain, and use it in combination with a benchtop cytometer to analyze and detect oral and oropharyngeal cancer cells ? first preclinically, and later with human tissues. In Specific Aim 2, we will use PARP Instastain to analyze fresh biopsy tissues. Using a handheld, portable fluorescent microscope, analyses will allow instantaneous diagnoses, first, in rat models and, second, in fresh human oral and oropharyngeal cancer tissues. If successful, the proposed novel technologies have the potential to improve and transform the current standard of care for oral and oropharyngeal cancer diagnosis, which would result in reduced health care costs and improved survival and quality of life for patients around the world.